The present invention is related to a method, a system, and a device for measuring particle settling velocity. In an exemplary embodiment, the invention is related to a method, a system, and a device for measuring the settling velocity of various particles in fields such as wastewater treatment, food processing, polymer and pigment technologies. The invention is particularly useful for measuring the settling velocity of biological entity such as cells in a suspension with a gravity settler e.g. a settling column, and can be widely applied in biotechnology and pharmaceutical fields, for example, to improve the efficiency of a perfusion cell culture process involving gravity settler for cell separation.
Accurate cell settling velocity determination is critical for perfusion culture using a gravity settler for cell retention. Gravity settlers have been successfully applied as cell retention devices in perfusion cell cultures from the bench-top to large-scale industrial applications, as disclosed in Batt, B.; Davis, R.; Kompala, D., Inclined sedimentation for selective retention of viable hybridomas in a continuous suspension bioreactor. Biotechnology Progress 1990, 6(6), 458-464; Choo, C. Y.; Tian, Y.; Kim, W. S.; Blatter, E.; Conary, J.; Brady, C. P., High-level production of a monoclonal antibody in murine myeloma cells by perfusion culture using a gravity settler. Biotechnol. Prog. 2007, 23(1), 225-231; Searles, J.; Todd, P.; Kompala, D., Viable cell recycle with an inclined settler in the perfusion culture of suspended recombinant chinese hamster ovary cells. Biotechnology Progress 1994, 10(2), 198-206; Wen, Z.-Y.; Teng, X.-W.; Chen, F., A novel perfusion system for animal cell cultures by two step sequential sedimentation. Journal of Biotechnology 2000, 79(1), 1-11; Shackel, I.; Bass, A.; Brewer, A.; Brown, P.; Tsao, M.; Chang, L., Comparison of manufacture technologies for rav12 monoclonal antibody in production medium containing no animal derived proteins. In Cell Technology for Cell Products, 2007; pp 761-763; Amazile B. R. A. Maia, D. L. N., Application of gravitational sedimentation to efficient cellular recycling in continuous alcoholic fermentation. Biotechnology and Bioengineering 1993, 41(3), 361-369; and Nottorf, T.; Hoera, W.; Buentemeyer, H.; Siwiora-Brenke, S.; Loa, A.; Lehmann, J., Production of human growth hormone in a mammalian cell high density perfusion process. In Cell Technology for Cell Products, 2007; pp 789-793.
The capacity of an inclined gravity settler to clarify cell suspension is described in equation (1):S(v)=v·sp  (1)where S(v) is the volumetric flow rate of fluid clarified of particles with sedimentation velocity v; sp is the projection area of an inclined gravity settler, given by w(L sin θ+b cos θ); w is the settler width, b is the separation between the two inclined surfaces, L is the length of the settler, and θ is the angle of inclination of the settler from the vertical. Batt et al. and Davis et al. have successfully predicted the cell retention efficiency of gravity settlers based on theoretically calculated cell settling velocities in Batt, B.; Davis, R.; Kompala, D., Inclined sedimentation for selective retention of viable hybridomas in a continuous suspension bioreactor, Biotechnology Progress 1990, 6(6), 458-464; and in Davis, R. H.; Lee, C. Y.; Batt, B. C.; Kompala., D. S., Cell separations using differential sedimentation in inclined settlers, in Cell separation science and technology, Dhinakar S. Kompala, P. T., Ed. 1991; pp 113-127.
As disclosed in Batt, B.; Davis, R.; Kompala, D., Inclined sedimentation for selective retention of viable hybridomas in a continuous suspension bioreactor, Biotechnology Progress 1990, 6(6), 458-464; and Searles, J.; Todd, P.; Kompala, D., Viable cell recycle with an inclined settler in the perfusion culture of suspended recombinant chinese hamster ovary cells. Biotechnology Progress 1994, 10(2), 198-206, the accurate determination of the viable cell sedimentation velocity is critical for controlling the operation of the gravity settler to minimize viable cell loss and maximize nonviable cell removal, and thus maximize viable cell concentration in the bioreactor. During long-term perfusion culture, the cell suspension is a mixture of viable and nonviable cells, and the nonviable cells have settling velocities that are less than that of the viable cells. Viable cell settling velocity can vary significantly among mammalian cell lines; for instance, the settling velocity of hybridoma cell line AB2-143.2 and CHO cell line M1-59 are 2.9 cm/hr and 1.45 cm/hr respectively, as disclosed in Searles, J.; Todd, P.; Kompala, D., Viable cell recycle with an inclined settler in the perfusion culture of suspended recombinant Chinese hamster ovary cells. Biotechnology Progress 1994, 10(2), 198-206. This two-fold difference demonstrates the necessity of measuring this parameter for every new cell line to be used in a gravity settler/perfusion system in order to properly select the gravity settler with appropriate capacity.
It is important to measure the distinct settling velocity of the viable and nonviable cell populations periodically during a long-term perfusion culture in order to optimize the operation of the gravity settler in real-time, because the settling velocity of viable cells may change substantially during the course of a long-term perfusion culture due to changes in cell size, as disclosed in Frame, K. K.; Hu, W.-S. Cell volume measurement as an estimation of mammalian cell biomass. Biotechnol. Bioeng. 1990, 36, 191-197; Martens D. E.; de Gooijer C. D.; van der Velden-de Groot C. A. M.; Beuvery E. C.; Tramper J. Effect of dilution rate on growth, productivity, cell cycle and size, and shear sensitivity of a hybridoma cell in a continuous culture. Biotechnol. Bioeng. 1993, 41, 429-439.
The measurement of erythrocyte sedimentation rate (ESR) has been widely used for over 50 years as a simple, standardized medical screening test, which has been disclosed in for example Council for Standardization in Haematology (Expert Panel on Blood Rheology), ICSH recommendations for measurement of erythrocyte sedimentation rate. J Clin Pathol. 1993, 46(3), 198-203; Woodland, N. B.; Cordatos, K.; Hung, W. T.; Reuben, A.; Holley, L., Erythrocyte sedimentation in columns and the significance of ESR. Biorheology 1996, 33(6), 477-488; Rabjohn, L.; Roberts, K.; Troiano, M.; Schoenhaus, H., Diagnostic and prognostic value of erythrocyte sedimentation rate in contiguous osteomyelitis of the foot and ankle. The journal of Foot and Ankle Surgery 2007, 46(4), 230-237; Mönig, H.; Marquardt, D.; Arendt, T.; Kloehn, S. Limited value of elevated erythrocyte sedimentation rate as an indicator of malignancy. Fam. Pract. 2002, 19, 436-438; Olshaker, J. S.; Jerrard, D. A., The erythrocyte sedimentation rate. Journal of Emergency Medicine 1997, 15(6), 869-874; and Erikssen, G.; Liestol, K.; Bjonholt, H.; Stormorken, H.; Thaulow, E.; Erikssen, J., Erythrocyte sedimentation rate: a possible marker of atherosclerosis and a strong predictor of coronary heart disease mortality. European Heart Journal 2000, 21(19), 1614-1620.
Many modifications have been made to speed-up the procedure, for example, Drucker, K. G. Sedimentation rate centrifuge and method determining sedimentation rate. U.S. Pat. No. 3,199,775, 1965; Winkelman, J. W.; Tanasijevic, M. J.; Bennett, M. Method and apparatus for determining erythrocyte sedimentation rate and hematocrit. U.S. Pat. No. 6,506,606, 2003; Bull, B. S. Method and apparatus for rapid determination of blood sedimentation rate. U.S. Pat. No. 5,594,164, 1997. However, the basic operational principle remains the same. A sample of blood is placed in a narrow tube (Westergren Tube) and after a period of time a visible interface forms between the clarified plasma and the red blood cells. By reading the scale at the interface after a defined period of time the sedimentation can be determined. This method assumes the red blood cells have uniform size and settling velocity; therefore the movement of the red blood cell population is taken as the distance that the cells at the top of the tube can move in certain time. This method is not directly applicable to mammalian cell culture, since there is not a clear color difference between the cells and the clarified supernatant. For the same reason, the method used to determine plant cell settling velocity is not practical for animal cell culture, as explained in De Dobbeleer, C.; Cloutier, M.; Fouilland, M.; Legros, R.; Jolicoeur, M., A high-rate perfusion bioreactor for plant cells. Biotechnology and Bioengineering 2006, 95(6), 1126-1137. Even if there is a clearly identifiable interface, only the settling velocity of the smallest nonviable cells can be determined in this manner. This measurement is much less important than that of the viable cells for optimizing the gravity settler operation.
Particle image velocimetry (PIV) has been used primarily for determining the settling velocity of individual particles in Guazzelli, É., Evolution of particle-velocity correlations in sedimentation, Physics of Fluids 2001, 13(6), 1537-1540. Despite the complexity of this process, it cannot distinguish between viable and nonviable cell settling velocity.
Another method, the “Owen Tube”, is a 1-L column used for determining the settling velocity of suspended particulate matter in natural body water, as disclosed in Dearnaley, M. P., Direct measurements of settling velocities in the owen tube: A comparison with gravimetric analysis. Journal of Sea Research 1996, 36(1-2), 41-47; Wolfstein, K., Fractionation and measurements of settling velocities of suspended matter using an owen tube. Journal of Sea Research 1996, 36(1-2), 147-152; and Puls, W.; Kühl, H., Settling velocity determination using the BIGDAN settling tube and the Owen settling tube. Journal of Sea Research 1996, 36(1-2), 119-125. Periodic samples are removed from the bottom of the Owen Tube and the dry weight measurement is used to determine the settling velocity. However, this method is not accurate for small sample amounts, the presence of cell debris would contribute to measurement error, and the process cannot distinguish the viability of the cells.
Stokes' law can be used to estimate the settling velocity of particles in fluid when the Reynold's number is less than 0.2, given by:
                    v        =                                            gd              p              2                        ⁡                          (                                                ρ                  p                                -                ρ                            )                                            18            ⁢                                                  ⁢            μ                                              (        2        )            where dp=particle diameter; μ=fluid viscosity; ρp=density of solid particle; ρ=density of carrying fluid; g is acceleration due to gravity. The particle diameter is normally determined by means of a Particle Size Analyzer (Particle Data Inc.) or Coulter Multisizer (Beckman Coulter, Fullerton, Calif.). The particle density is measured using neutral buoyancy measurement or density gradient partitioning methods. A glass capillary viscometer can be used to determine the fluid viscosity. The fluid density is easily determined from weight and volume measurements. Using this procedure, the settling velocity of viable and nonviable hybridoma and CHO cells have been determined in Batt, B.; Davis, R.; Kompala, D., Inclined sedimentation for selective retention of viable hybridomas in a continuous suspension bioreactor. Biotechnology Progress 1990, 6(6), 458-464; and Searles, J.; Todd, P.; Kompala, D., Viable cell recycle with an inclined settler in the perfusion culture of suspended recombinant chinese hamster ovary cells. Biotechnology Progress 1994, 10(2), 198-206. However, this method is not practical for routine measurements during long-term perfusion culture since multiple measurements are needed for a single settling velocity determination, which is time-consuming and increases the potential for measurement error.
Advantageously, the present invention provides a simple, inexpensive, accurate, and rapid method for measuring settling velocity of particles such as polystyrene, and viable and nonviable cells in a mixed population, among others.